1. Field of the Invention
The present invention is broadly concerned with methods and compositions useful for detecting and identifying species of Candida. More particularly, the present invention relates to methods in which oligonucleotides are used as forward and reverse primers in polymerase chain reactions using nucleic acids from biological samples as templates, as probes for detecting any resultant amplicon, and as nucleotide sequencing primers to determine whether an resultant amplicon is specific to Candida ablicans, Candida glabrata, Candida parapsilosis, or Candida tropicalis, thereby allowing one to ascertain whether the samples contains any of these isolates.
2. Description of the Related Art
Vaginal candidiasis causes 20% to 25% of infectious vaginitis cases, second only to the 40% to 50% of cases caused by bacterial vaginosis (see Sobel, 1997, Vaginitis, N Engl J Med 337:1896-1903). Candida vaginitis (CV) is marked by pruritis, soreness, a change in discharge, dyspareunia, vulvar erythema, edema and fissures (see Sobel 1997, Vaginitis, N Engl J Med 337:1896-1903; and Diagnosis of Candidia vaginitis, 1985, J Fam Pract 20(1):19-20). The condition is rate before puberty, but by the age of 25, nearly one-half of all women will have had at least on clinician-diagnosed episode of CV. Overall, it is estimated that 75% of women will experience an episode of CV in their lifetime (see Sobel 1997, Vaginitis, N Engl J Med 337:1896-1903; and Sobel, 1988, Pathogenesis and epidemiology of vulvovaginal candidiasis, Ann N Y Acad Sci 544:547-557). Among the Candida species causing infections, Candida ablicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis account for 80% to 90% of fungal isolates encourntered worlwide (see Pfaller, 1996, Nosocomial candidiasis: emerging species, reservoirs, and modes of transmission, Clin Infect Dis 22(2):S89-S94; and Hazen et al., 2003, Comparison of the susceptibilities of Candida spp. to fluconazole and voriconazole in a 4-year global evaluation using disk diffusion, J Clin Microbiol 41:5623-5632). Although Candida albicans is implicated in 85% to 95% of all cases of CV (see Sobel, 1997, Vaginitis, N Engl J Med 337:1896-1903; and Sobel, 1999, Vulvovaginitis in healthy women, Compr Ther 25:335-346), the widespread use of azole antifungal drugs is postulated to have promoted the shifting of vaginal colonization and selection of more naturally resistant species, such as Candida glabrata (see Pfaller et al., 2003, Activities of fluconazole and voriconazole against 1,586 recent clinical isolates of Candida species determined by Broth microdilution, disk diffusion, and Etest methods: report from the ARTEMIS Global Antifungal Susceptibility Program, 2001 J Clin Microbiol 41:1440-1446; Snydman, 2003, Shifting patterns in the epidemiology of nosocomial Candida infections, Chest 123:500 S-503S; Hazen, 1995, New and emerging yeast pathogens, Clin Microbiol Rev 8:462-478; and Fidel et al., 1999, Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans, Clin Microbiol Rev 12:80-96). Knowledge of the infecting species is highly predictive of likely drug susceptibility and should be used as a guide for selecting therapy (see Pappas et al., 2004, Guidelines for treatment of candidiasis, Clin Infect Dis 38:161-189). Therefore, rapid and specific identification of Candida species would facilitate appropriate antifungal selection and improve patient care.
Commonly, Candida in vaginal samples is identified by microscopic examination of a wet mount with potassium hydroxide. This technique detects budding yeast cells in only 50% to 70% of women with CV (see Elliott, 1998, Managing patients with vulvovaginal candidiasis, Nurse Pract 23:44-46, 49-53; and 1996, ACOG technical bulletin. Vaginitis. Number 226-July 1996 (replaces No. 221, March 1996). Committee on Technical Bulletins of the American College of Obstetricians and Gynecologists, Int J Gynaecol Obstet 54:293-302) and may fail to detect species other than Candida albicans (see 1996, ACOG technical bulletin. Vaginitis. Number 221-March 1996 (replaces no. 135, November 1989). American College of Obstetricians and Gynecologists, Int J Gynaecol Obstet 53:271-280). Alternatively, Candida albicans and Candida tropicalis can be distinguished by growth on chromogenic agar medium and other species of Candida can be identified by enzymatic tests. However, each of these tests requires isolated organisms to be grown on solid medium for 24 to 48 hours before they can be performed or interpreted (see Odds et al., 1994, CHROMagar Candida, a new differential isolation medium for presumptive identification of clinically important Candida species, J Clin Microbiol 32:1923-1929; and Warren et al., 1995, Cryptococcus, and other yeasts of medical importance, in: P. R. Murray, E. J. Barton, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (Eds.), Manual of clinical microbiology, American Society for Microbiology, Washington, D.C., pp. 723-737). In addition, the “gold standard” for definitive biochemical identification requires analysis of assimilation and fermentation, taking up to 30 days to complete (see Warren et al., 1995, Cryptococcus, and other yeasts of medical importance, in: P. R. Murray, E. J. Barton, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (Eds.), Manual of clinical microbiology, American Society for Microbiology, Washington, D.C., pp. 723-737).
In recent years, numerous DNA-based techniques have been developed to improve the identification of Candida species. Amplification of Candida target DNA by the polymerase chain reaction (PCR) is particularly promising because of its simplicity, specificity, and sensitivity (see Chen et al., 2000, Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes, J Clin Microbiol 38:2302-2310; Luo et al., 2002, Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR, J Clin Microbiol 40:2860-2865; Evertsson et al., 2000, Detection and identification of fungi in blood using broad-range 28S rDNA PCR amplification and species-specific hybridisation, Apmis 108:385-392; Tamura et al., 2000, New PCR primer pairs specific for Candida dubliniensis and detection of the fungi from the Candida albicans clinical isolates in Japan, Clin Lab 46:33-40; Wahyuningsih et al., 2000, Simple and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis, J Clin Microbiol 38:3016-3021; and Elie et al., 1998, Rapid identification of Candida species with species-specific DNA probes, J Clin Microbiol 36:3260-3265). However, these strategies require post-amplification analyses and are considered to have lower sensitivity than real-time PCR techniques that directly monitor amplification via fluorescent probes (see Holland et al., 1991, Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase, Proc Natl Acad Sci USA 88:7276-7280). Real-time PCR strategies have been developed to identify Candida species (see Guiver et al., 2001, Rapid identification of Candida species by TaqMan PCR, J Clin Pathol 54:362-366; Borst et al., 2001, Detection of Candida spp. in blood cultures using nucleic acid sequence-based amplification (NASBA), Diagn Microbiol Infect Dis 39:155-160; Shin et al., 1999, Rapid identification of up to three Candida species in a single reaction tube by a 5′ exonuclease assay using fluorescent DNA probes, J Clin Microbiol 37:165-170; and Selvarangan et al., 2003, Rapid identification of commonly encountered Candida species directly from blood culture bottles, J Clin Microbiol 41:5660-5664), but these methods were designed and optimized for detection of Candida in blood or blood culture. Strategies for the detection of Candida species in DNA extracted from vaginal samples, especially without time-consuming culture, are lacking. In addition, current DNA-based Candida detection methods do not take into account the fact that DNA sequencing is generally accepted as the most precise method for discriminating among closely related species.